The present invention relates to methods, compositions and devices for identifying microorganisms. More specifically, the invention relates to the use of a matrix of polynucleotide probes having specificity for ribosomal nucleic acids (rRNA and rDNA) that distinguish species or groups of organisms from each other based on molecular phylogeny.
The procedures for detecting and identifying infectious organisms are some of the most critical tasks performed in the clinical laboratory. Whereas laboratory diagnoses of infectious diseases formerly were made by experienced technicians using visual inspection of stained clinical material, more rapid and objective results are obtainable using modern techniques. Immunoassays, including radioimmunoassays, enzyme-linked immunoassays, and latex agglutination and immunoblotting assays have developed into powerful diagnostic tools having utilities that are enhanced by the availability of monoclonal antibodies. More recent advances in signal and target amplification have introduced the era of molecular diagnostics based on the use of polynucleotide probes.
Indeed, clinical microbiologists now use an extensive array of techniques for identifying infectious organisms (see Manual of Clinical Microbiology Murray et al., eds., 6th edition, ASM Press (1995)). Automated substrate utilization systems typically rely on enzymatic reactions that release chromogenic of fluorogenic compounds, tetrazolium-based indicators of metabolic activity in the presence of different carbon sources, or detection of the acid products of metabolism. The patterns of positive and negative reactions with these substrates establish a biochemical profile that can be used to identify microorganisms isolated from clinical samples. The chromatographic profiles of the more than 300 fatty acids that contribute to the formation of lipids in bacteria and yeast have also been used to phenotype microorganisms. Despite the availability of these very powerful techniques, polynucleotide-based assays are rapidly gaining popularity in clinical laboratory practice.
The specificity of polynucleotide hybridization reactions, together with the extraordinary sensitivity afforded by nucleic acid amplification techniques, has made molecular diagnostics the method of choice for detecting and identifying microbes that are available in only very small quantities. Commonly used DNA probe hybridization formats include: solid phase hybridization, solution-phase hybridization and in situ hybridization. In solid phase hybridization methods, a sample containing microbial polynucleotides is immobilized to a solid support, denatured and then probed with a polynucleotide probe that harbors a detectable label. Unhybridized probe is removed from the system and specifically hybridized probe detected, for example by autoradiography or direct visual observation. In solution-phase hybridization procedures, the target polynucleotide and the labeled probe are free to interact in an aqueous hybridization buffer. Specifically hybridized probe is then detected as an indicator of the presence of target polynucleotides in the mixture. In situ hybridization using formalin-fixed tissue sections is used for obtaining information about the physical distribution and abundance of microorganisms. As they are conventionally practiced, molecular diagnostic assays are conducted to determine whether a particular species or group of organisms is present in a biological sample undergoing testing.
Bacteremia and fungemia are conditions marked by the presence of bacterial and fungal organisms in circulating blood. Sepsis refers to a severe bacterial infection that spreads in the blood throughout the entire body. In septicemia, the presence of microorganisms in the blood indicates that the host""s immune system has failed to localize an infection. Organisms responsible for these conditions typically are identified after inoculating a xe2x80x9cblood culture bottlexe2x80x9d with a sample of patient blood, and then typing any microorganisms that are propagated. Mortality associated with bloodstream infections ranges from an estimated 20% to 50% (see Clinics in Laboratory Medicine 14:9 (1994)). An estimated 50,000 deaths each year in the United States result from sepsis (Vanderbilt Univ. Med. Center Reporter 1991 Mar. 1; 2(8):1,3). Thus, substantial attention has been devoted to the diagnosis and treatment of this syndrome.
Unfortunately, the blood culture methods that represent the xe2x80x9cgold standardxe2x80x9d for diagnosing septicemia have significant limitations (Weinstein, Clin Infect Dis 23:40 (1996)). Indeed, no single medium is ideal for propagating all potential bloodstream organisms, some relevant microorganisms grow poorly in conventional media and systems, and positive results require hours to days of incubation. Each of these areas calls for improvement if diagnosis of septicemia is to become more rapid and accurate.
Molecular diagnostic methods employing collections of species-specific DNA probes have been used to identify microorganisms in blood cultures. According to one procedure, microorganisms present in growth-positive culture bottles were first isolated and then Gram-stained and analyzed for morphology (Davis et al., J. Clin Microbiol. 29:2193 (1991)). The appearance of the stained organisms determined which of several different polynucleotide probes were employed in a subsequent testing step. Instances wherein positive hybridization results were obtained yielded presumptive identifications. Identification of bacteria that yielded negative hybridization results were limited to observations made after Gram-staining and microscopic analysis. These investigations confirmed that DNA probes could be used for the rapid identification of bacteria taken directly from blood culture bottles, but still required evaluation of Gram-stained microorganisms by an experienced microbiologist. Importantly, polymicrobial bacteremias could not be analyzed in the procedure due to the lack of available probes.
In a study of several hundred positive blood cultures obtained from septicemia patients, Weinstein et al., in Clin Infect Dis 24:584 (1997) concluded that the five most common pathogens were Staphylococcus aureus, Escherichia coli, coagulase-negative staphylococci, Klebsiella pneumoniae, and Enterococcus species. Yeasts were also common isolates from blood culture bottles and represented true fungemia when detected about 92% of the time. Candida albicans ranked among the top 10 microorganisms causing septicemia. Interestingly, about 91% of the instances of bacteremia and fungemia were unimicrobial while the remaining cases were associated with two or three organisms. The lowest associated mortality among the patients having positive blood cultures were associated with coagulase-negative staphylococci (5.5%) and the highest with yeasts and fungi (35.8%). Most important, septicemia-associated mortality was shown to increase in proportion to the duration of inappropriate antimicrobial therapy. This finding highlighted the importance of an early and proper clinical diagnosis.
The invention detailed below provides a rapid means for identifying microorganisms using techniques that are suited for automated analysis. The invented devices and methods can even be used to resolve the identity of microorganisms that are contained in a mixed population of microorganisms.
One aspect of the present invention relates to a device for hybridizing nucleic acids. The invented device includes a solid support and a plurality of addresses disposed on the solid support. Each of the addresses includes at least one probe that hybridizes ribosomal nucleic acids from at least one microbial species under high stringency hybridization conditions. The plurality of addresses includes a higher-order address, an intermediate-order address, and a lower-order address. The lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the intermediate-order address. The intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address. In certain embodiments the solid support of the device is either multiwell plate or a plurality of individual tubes that are maintained in a spaced-apart configuration. In certain preferred embodiments the higher-order address is a pan-bacterial address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram(+) bacteria, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus Enterococcus, a plurality of species of bacteria in the genus Staphylococcus, and a plurality of species of bacteria in the genus Campylobacter. In alternative embodiments the higher-order address is a pan-fungal address that specifically hybridizes ribosomal nucleic acids from a plurality of fungal species. Certain embodiments of the invented device include both a pan-bacterial address as the higher-order address, and a pan-fungal address. The higher-order address can also be a Gram(+) address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram(+) bacteria. When the higher-order address is a pan-bacterial address, and regardless of whether or not there is included an address for detecting pan-fungal organisms, the intermediate-order address may be a Gram(+) address that specifically hybridizes ribosomal nucleic acids of a plurality of Gram(+) bacteria, a family Enterobacteriaceae address that specifically hybridizes ribosomal nucleic acids from a plurality of bacteria in the family Enterobacteriaceae, a Staphylococcus genus address that specifically hybridizes ribosomal nucleic acids from a plurality of species in the Staphylococcus genus, a genus Enterococcus address that specifically hybridizes ribosomal nucleic acids from a plurality of species in the genus Enterococcus or a Campylobacter address that specifically hybridizes ribosomal nucleic acids from a plurality of Campylobacter species. In a preferred embodiment the intermediate-order address is the Gram(+) address and the lower-order address is an Actinomycetes address that specifically hybridizes ribosomal nucleic acids of a plurality of bacteria belonging to the High (G+C) subset of Gram(xe2x88x92) bacteria. In another preferred embodiment the intermediate-order address is the Gram(+) address and the lower-order address is an address that specifically hybridizes ribosomal nucleic acids from a plurality of Mycobacterium species. For example, the plurality of Mycobacterium species may include Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG and Mycobacterium africanum. In still another preferred embodiment when the higher-order address is a pan-bacterial address, and regardless of whether or not there is included an address for detecting pan-fungal organisms, the intermediate-order address is the Gram(xe2x88x92) address and the lower-order address is an address that specifically hybridizes ribosomal nucleic acids of Streptococcus pneumoniae. In yet another preferred embodiment when the higher-order address is a pan-bacterial address, and regardless of whether or not there is included an address for detecting pan-fungal organisms, the intermediate-order address is the Gram(xe2x88x92) address and the lower-order address is an address that specifically hybridizes ribosomal nucleic acids from Listeria monocytogenes. In still yet another preferred embodiment of the invention when the higher-order address is a pan-bacterial address, and regardless of whether or not there is included an address for detecting pan-fungal organisms, the intermediate-order address is the Gram(+) address, and the lower-order address is an address that specifically hybridizes ribosomal nucleic acids of Staphylococcus aureus. A different device according to the invention includes a pan-bacterial address as the higher-order address and, regardless of whether or not there is included an address for detecting pan-fungal organisms, the intermediate-order address is the family Enterobacteriaceae address and the lower-order address is an E. coli address that specifically hybridizes ribosomal nucleic acids of E. coli. In another preferred embodiment when the higher-order address is a pan-bacterial address, and regardless of whether or not there is included an address for detecting pan-fungal organisms, the lower-order address is a Staphylococcus aureus address that specifically hybridizes ribosomal nucleic acids from Staphylococcus aureus. In another preferred embodiment when the higher-order address is a pan-bacterial address, and regardless of whether or not there is all included an address for detecting pan-fungal organisms, the intermediate-order address is the genus Enterococcus address. In another preferred embodiment when the higher-order address is a pan-bacterial address, and regardless of whether or not there is included an address for detecting pan-fungal organisms, the intermediate-order address is the Campylobacter address. In other preferred devices the higher-order address is the Gram(+) a address and the lower-order address is an address that detects ribosomal nucleic acids from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG and Mycobacterium africanum. In still other devices the higher-order address is a pan-fungal address, the intermediate-order address specifically hybridizes ribosomal nucleic acids from a plurality of Candida species including Candida albicans, Candida tropicalis, Candida dubliniensis, Candida viswanathii and Candida parapsilosis and the lower-order address specifically hybridizes ribosomal nucleic acids from Candida albicans and Candida dubliniensis but not Candida tropicalis, Candida viswanathii or Candida parapsilosis. In another preferred embodiment when the higher-order address is a pan-bacterial address, there is included a pan-fungal address that hybridizes ribosomal nucleic acids from a plurality of fungal species. In a highly preferred embodiment the device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address and an Actinomycetes address. An even more highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address and an addresses that hybridizes ribosomal nucleic acids from bacteria in the family Enterobacteriaceae. An alternative highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address and an addresses that hybridizes ribosomal nucleic acids from Enterococcus bacteria. In devices having a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address and an addresses that hybridizes ribosomal nucleic acids from bacteria in the family Enterobacteriaceae, there can be further included an address that hybridizes ribosomal nucleic acids from Enterococcus bacteria. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address and a single address that hybridizes ribosomal nucleic acids of Enterococcus bacteria and ribosomal nucleic acids from bacteria in the family Enterobacteriaceae. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address and an address that hybridizes ribosomal nucleic acids from bacteria in the Staphylococcus genus. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address and an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus Campylobacter. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address, an address that hybridizes ribosomal nucleic acids from bacteria in the Staphylococcus genus, and an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus Campylobacter. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(xe2x88x92) address, an Actinomycetes address, an address that hybridizes ribosomal nucleic acids from bacteria in the family Enterobacteriaceae, an address that hybridizes ribosomal nucleic acids of Enterococcus bacteria, and further includes an address that hybridizes ribosomal nucleic acids from bacteria in the Staphylococcus genus and an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus Campylobacter. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(xe2x88x92) address, an Actinomycetes address and a single address that hybridizes ribosomal nucleic acids of Enterococcus bacteria and ribosomal nucleic acids from bacteria in the family Enterobacteriaceae, as well as a single address that hybridizes ribosomal nucleic acids from bacteria in the Staphylococcus genus and ribosomal nucleic acids from a plurality of bacteria in the genus Campylobacter. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address, an Actinomycetes address, an address that hybridizes ribosomal nucleic acids from bacteria in the family Enterobacteriaceae, an address that hybridizes ribosomal nucleic acids of Enterococcus bacteria, and further includes an address that hybridizes ribosomal nucleic acids from bacteria in the Staphylococcus genus and an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus Campylobacter, and further includes at least one address that specifically hybridizes ribosomal nucleic acids from a single microorganism species. In a highly preferred embodiment the single microorganism species is selected from the group consisting of Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, Candida albicans and Staphylococcus aureus. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(xe2x88x92) address, an Actinomycetes address, and a single address that hybridizes ribosomal nucleic acids of Enterococcus bacteria and ribosomal nucleic acids from bacteria in the family Enterobacteriaceae, as well as a single address that hybridizes ribosomal nucleic acids from bacteria in the Staphylococcus genus and ribosomal nucleic acids from a plurality of bacteria in the genus Campylobacter, as well as a plurality of addresses that individually hybridize ribosomal nucleic acids from a plurality of microorganism species. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address and an Actinomycetes address, wherein the pan-bacterial address includes a polynucleotide probe having a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:58. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address and an Actinomycetes address, wherein the pan-fungal address comprises a polynucleotide probe having the sequence of SEQ ID NO:4. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address and an Actinomycetes address, wherein the Gram(+) address includes a polynucleotide probe having the sequence of SEQ ID NO:7. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address and an Actinomycetes address, wherein the Actinomycetes address includes a polynucleotide probe having the sequence of SEQ ID NO: 10. Yet another highly preferred device includes a pan-fungal address, a pan-bacterial address, a Gram(+) address and an Actinomycetes address, wherein the pan-bacterial address includes a polynucleotide probe having a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:58, wherein the pan-fungal address includes a polynucleotide probe having the sequence of SEQ ID NO:4, wherein the Gram(+) address includes a polynucleotide probe having the sequence of SEQ ID NO:7, and wherein the Actinomycetes address includes a polynucleotide probe having the sequence of SEQ ID NO: 10. Still yet another highly preferred embodiment of the invented device has each of the probes labeled with an acridinium ester.
Another aspect of the invention relates to a method of analyzing a biological sample suspected of containing microorganisms. The method begins with a step for obtaining the biological sample. Thereafter there is a step for culturing the biological sample for a period of time sufficient to increase in number any microorganisms contained in the sample. Next, there is a step for releasing polynucleotides from any microorganisms in the cultured biological sample, and then hybridizing the released polynucleotides with a probe matrix. According to some embodiments of the invented method the released polynucleotides have not been amplified in an in vitro polynucleotide amplification reaction. Other embodiments provide for the amplification of polynucleotides, for example by in vitro amplification, prior to the hybridizing step. The polynucleotide probe matrix includes a plurality of addresses with each address including at least one probe that hybridizes ribosomal nucleic acids from one or more microbial species under high stringency conditions. The plurality of addresses includes: a higher-order address, an intermediate-order address, and a lower-order address. The lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the intermediate-order address. The intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address. Additional steps in the invented method involve detecting positive and negative hybridization results for each of said plurality of addresses to establish a hybridization profile; and then comparing the hybridization profile with a look-up table that correlates the identities of microorganisms with hybridization results at each of the addresses. This procedure provides information about the identity of microorganisms contained in the biological sample. In a preferred embodiment, the salt concentration in the hybridization reaction can be in the range of from 0.6-0.9 M when the hybridization temperature is in the range of from 55-65xc2x0 C. Regardless of the choice of high stringency conditions that are selected, the higher-order address in the hybridizing step may be a pan-bacterial address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram(+) bacteria, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus Enterococcus, a plurality of species of bacteria in the genus Staphylococcus, and a plurality of species of bacteria in the genus Campylobacter, and the plurality of addresses in the hybridizing step may include a pan-fungal address that specifically hybridizes ribosomal nucleic acids from a plurality of fungal species. When this is the case, and in a preferred embodiment, the intermediate-order address in the hybridizing step can be Gram(+) address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram(+) bacteria, and the lower-order address in the hybridizing step can be an Actinomycetes address that specifically hybridizes ribosomal nucleic acids of a plurality of bacteria belonging to the High (G+C) subset of Gram(+) bacteria. Optionally, the biological sample in the obtaining step can be a sample of blood drawn from an individual being tested for a medical condition selected from the group consisting of bacteremia, septicemia and fungemia, and the culturing step may include a step for inoculating a blood bottle with an aliquot of the sample of blood and thereafter incubating the inoculated blood bottle. In a highly preferred embodiment, the detecting step involves detecting by luminometry.
Still another aspect of the invention relates to a method of analyzing a sample containing ribosomal nucleic acids. This method begins with a step for hybridizing the sample with a probe matrix under high stringency conditions to result in a hybridized sample. The probe matrix has a plurality of addresses, including a higher-order address, an intermediate-order address, and a lower-order address. The lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the intermediate-order address. The intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address. Another step in the invented method involves analyzing the hybridized sample to identify a first collection of addresses having at least one probe complementary to ribosomal nucleic acids present in the sample and to identify a second collection of addresses not having at least one probe complementary to ribosomal nucleic acids present in said sample. Thereafter there is a step for determining from the first and second collections of addresses identified in the analyzing step which of a collection of microorganisms possess ribosomal nucleic acids having a corresponding profile of ribosomal nucleic acid sequences, thereby determining the microbial origin of the RNA containing sample. In a preferred embodiment the higher-order address in the hybridizing step is a pan-bacterial address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram(+) bacteria, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus Enterococcus, a plurality of species of bacteria in the genus Staphylococcus, a and a plurality of species of bacteria in the genus Campylobacter, and the plurality of addresses in the hybridizing step further includes a pan-fungal address that specifically hybridizes ribosomal nucleic acids from a plurality of fungal species. When this is the case the intermediate-order address in the hybridizing step can be a Gram(+) address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram(+) bacteria, and the lower-order address in the hybridizing step can be an Actinomycetes address that specifically hybridizes ribosomal nucleic acids of a plurality of bacteria belonging to the High (G+C) subset of Gram(+) bacteria. The analyzing step in the invented method may involve analyzing by luminometry. Optionally the hybridizing step is conducted between 55xc2x0 C. and 65xc2x0 C.
Still yet another aspect of the invention relates to a device that is used for analyzing results from a probe matrix hybridization procedure. This analytical device includes a memory device having stored therein a look-up table. This look-up table correlates the identities of a plurality of microorganisms with positive and negative hybridization results at a plurality of addresses in a probe matrix. The plurality of addresses includes at least a higher-order address, an intermediate-order address, and a lower-order address. The lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the intermediate-order address. The intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address. The device also includes a processor linked to the memory device, where the processor is configured to execute a comparison between results inputted into the processor and the look-up table. These results will indicate positive and negative hybridization at the plurality of addresses in the probe matrix for polynucleotides released from the microorganism. The device further includes a user interface linked to the processor for initiating the comparison and an output device linked to the processor for displaying the results of said comparison. In a preferred embodiment the memory device includes magnetic storage media. In a different preferred embodiment the user interface includes a keyboard for inputting into the processor the positive and negative hybridization results. In yet another preferred embodiment the analytical device further includes a probe hybridization detector linked to the processor through an interface for inputting into the processor said positive and negative hybridization results. When this is the case, the detector can include a luminometer. In a highly preferred embodiment the output device is a visual display monitor or a printer.
Still yet another aspect of the invention relates to a method of determining whether a sample of bacteria includes a Staphylococcal species other than Staphylococcus aureus. This method begins with a step for releasing polynucleotides from the sample of bacteria to result in a collection of released polynucleotides. Subsequently there is a step for hybridizing the collection of released polynucleotides with a first probe having specificity for ribosomal nucleic acids of bacteria in the genus Staphylococcus and with a second probe having specificity for ribosomal nucleic acids of Staphylococcus aureus. Thereafter there is a step for detecting any of the first probe or the second probe that hybridized the collection of released polynucleotides. Finally, there is a step for determining that the sample of bacteria contains a Staphylococcal species other than Staphylococcus aureus if the first probe hybridized and the second probe did not hybridize.